Bioengineered corneal endothelium is being explored as an exciting, practical alternative to corneal transplantation to treat vision loss due to endothelial injury or disease. Currently, donor human corneal endothelial cells (HCEC) are being used to develop bioengineered constructs;however, HCEC have a finite ability to divide, thus limiting the number of healthy cells for use in these constructs. Our goal is to develop a novel approach to ensure an available supply of healthy HCEC for bioengineered corneal constructs and tissue repair. The proposed exploratory studies are based on the fact that mesenchymal stem cells from several sources, including human umbilical cord blood, have the potential to differentiate into several cell types. Since HCEC differentiate from neural crest-derived mesenchymal cells during ocular development, it is reasonable to hypothesize that human umbilical cord blood mesenchymal stem cells (UCB MSCs) can be differentiated into functional HCEC by recapitulating microenvironmental conditions that contribute to the development of corneal endothelium from neural crest-derived mesenchyme. We propose three Specific Aims to test this hypothesis. Aim 1 will establish a set of baseline characteristics for MSCs and HCEC that can be used to follow MSC differentiation to HCEC-like cells. Microarray analysis will determine relative gene expression, flow cytometry will identify surface marker expression, Western blots will compare expression of specific cellular and matrix proteins. Immunostaining for Ki67 and ZO-1 will test for contact inhibition of proliferation. Transmission electron microscopy will examine cellular morphology. Aim 2 will identify culture conditions that differentiate UCB MSCs to HCEC-like cells. Culture conditions will try to recapitulate the microenvironment that leads to formation of mature HCEC during eye development. UCB MSCs will be seeded on 4 different matrices and successively cultured in lens epithelial cell-conditioned medium or co-cultured with lens epithelial cells;cultured in HCEC- conditioned medium or co-cultured with HCEC;and then cultured in aqueous humor. The effect of these culture conditions on MSCs will be followed by identifying changes in characteristics from those exhibited by undifferentiated MSCs to those exhibited by HCEC as in Aim 1. Aim 3 will test the function of differentiated HCEC-like MSCs using an in vivo mouse model. Differentiated, HCEC-like MSCs will be seeded onto donor mouse corneas that have been denuded of endothelial cells. This corneal construct will be transplanted into a recipient mouse eye. The contralateral eye, corneal transplants containing cultured HCEC, and donor corneas with normal mouse endothelium will act as controls. The ability of the differentiated MSCs to maintain a clear cornea will be followed in vivo over an 8-week period by scoring for corneal opacity and vessel in-growth. Corneas will also be removed at weekly intervals to monitor cell size, shape, and number, and to test for maintenance of a contact-inhibited monolayer. Successful completion of these exploratory studies could have a major impact on the treatment of corneal disease by providing a new, healthy source of HCEC-like cells to treat patients who have lost visual acuity due to dysfunction of this physiologically important tissue. PUBLIC HEALTH RELEVANCE When corneal endothelial cells die due to injury or disease, they do not divide to replace themselves, resulting in loss of corneal clarity and visual acuity. Successful completion of these exploratory studies to test whether human umbilical cord blood mesenchymal stem cells can be differentiated to form corneal endothelial cells could have a major impact on the treatment of corneal disease by providing a new, healthy source of endothelial cells to treat patients who have lost corneal clarity and visual acuity due to dysfunction of this physiologically important tissue.